This invention is related to compositions and methods for decreasing the cholesterol levels in a subject. More specifically, the invention pertains to delivering a nucleic acid expression constrict that encodes growth-hormone-releasing-hormone (“GHRH”) into a tissue of the subject, wherein, GHRH is expressed in vivo in the subject, and has the effect of decreasing the cholesterol levels in that subject. The subject for this invention can be a human, pig, cow, bird or any other animal species.
High cholesterol level remains a significant problem in both humans and animals. Recent data from the American Medical Association shows that 30% of the entire population of the US is obese, including children, and at risk at developing pathologies induced in part by high cholesterol levels such as heart disease. Thus, cholesterol-decreasing therapies are required. Substantial efforts have addressed the prevention rather than treatment of disease. Hypothalamic GHRH stimulates growth hormone (“GH”) secretion from the anterior pituitary gland, but recent studies have also demonstrated other properties of this peptide (Siejka et al., 2004).
Cholesterol. Cholesterol is a sterol (a combination steroid and alcohol) and a lipid found in the cell membranes of all body tissues, and transported in the blood plasma of all animals. Lesser amounts of cholesterol are also found in plant membranes.
Most cholesterol in animals is NOT dietary in origin and is synthesized internally. Cholesterol is present in higher concentrations in tissues which either produce more or have more densely-packed membranes, for example, the liver, spinal cord and brain, and also in atheromas. Cholesterol plays a central role in many biochemical processes, but is best known for the association of cardiovascular disease with various lipoprotein cholesterol transport patterns and high levels of cholesterol in the blood.
Often, when most doctors talk to their patients about the health concerns of cholesterol, they are referring to “bad cholesterol”, or low-density lipoprotein (LDL). “Good cholesterol” is high-density lipoprotein (HDL).
Cholesterol is required to build and maintain cell membranes and makes the membrane's fluidity stable over wider temperature intervals. This is possible due to the hydroxyl group on cholesterol that interacts with the phosphate head of the membrane, and the bulky steroid and the hydrocarbon chain being embedded in the membrane. Some research indicates that cholesterol may act as an antioxidant. Cholesterol also aids in the manufacture of bile (which helps digest fats), and is also important for the metabolism of fat soluble vitamins, including vitamins A, D, E and K. Cholesterol is the major precursor for the synthesis of vitamin D, of the various steroid hormones, including cortisol and aldosterone in the adrenal glands, and of the sex hormones progesterone, estrogen, and testosterone. Further recent research shows that cholesterol has an important role for the brain synapses as well as in the immune system, including protecting against cancer.
Recently, cholesterol has also been implicated in cell signaling processes, where it has been suggested that it forms lipid rafts in the plasma membrane. It also reduces the permeability of the plasma membrane to proton and sodium ions. Cholesterol is minimally soluble in water, therefore, it cannot dissolve and travel in the water-based bloodstream. Instead, cholesterol is transported in the bloodstream by lipoproteins that are water-soluble and carry cholesterol and fats internally. The proteins forming the surface of the given lipoprotein particle determine from what cells cholesterol will be removed and to where it will be supplied.
The largest lipoproteins are called chylomicrons, and function to primarily transport fats from the intestinal mucosa to the liver. They carry mostly triglyceride fats and cholesterol. In the liver, chylomicron particles give up triglycerides and some cholesterol, and are converted into low-density lipoprotein (LDL) particles, which carry triglycerides and cholesterol on to other body cells. In healthy individuals the LDL particles are large and relatively few in number. In contrast, large numbers of small LDL particles are strongly associated with promoting disease within the arteries.
High-density lipoprotein (HDL) particles transport cholesterol back to the liver for excretion, but vary considerably in their effectiveness for doing this. Having large numbers of large HDL particles correlates with better health outcomes. In contrast, having small amounts of large HDL particles is strongly associated with disease progression within the arteries.
The cholesterol molecules present in LDL cholesterol and HDL cholesterol are identical. The difference between the two types of cholesterol derives from the carrier protein molecules or the lipoprotein component.
Biosynthesis of cholesterol is directly regulated by the cholesterol levels present, though the homeostatic mechanisms involved are only partly understood. A higher intake from food leads to a net decrease in endogenous production, while lower intake from food has the opposite effect. Although not wanting to be bound by theory, the main regulatory mechanism is the sensing of intracellular cholesterol in the endoplasmic reticulum by the protein SREBP (Sterol Regulatory Element Binding Protein 1 and 2). In the presence of cholesterol, SREBP is bound to two other proteins: SCAP (SREBP-cleavage activating protein) and Insig-1. When cholesterol levels fall, Insig-1 dissociates from the SREBP-SCAP complex, allowing the complex to migrate to the Golgi apparatus, where SREBP is cleaved by S1P and S2P (site 1/2 protease), two enzymes that are activated by SCAP when cholesterol levels are low. The cleaved SREBP then migrates to the nucleus and acts as a transcription factor to bind to the SRE (sterol regulatory element) of a number of genes to stimulate their transcription. Among the genes transcribed are the LDL receptor and HMG-CoA reductase. The former scavenges circulating LDL from the bloodstream, whereas HMG-CoA reductase leads to an increase of endogenous production of cholesterol. An excess of cholesterol in the bloodstream may lead to its accumulation in the walls of arteries. This build up is what can lead to clogged arteries and eventually to heart attacks and strokes.
The average amount of blood cholesterol varies with age, typically rising gradually until one is about 60 years old. There appear to be seasonal variations in cholesterol levels in humans, more, on average, in winter.
Cholesterol is excreted from the liver in bile and reabsorbed from the intestines. Under certain circumstances, when more concentrated, as in the gallbladder, it crystallises and is the major constituent of most gallstones, although lecithin and bilirubin gallstones also occur less frequently.
Growth hormone (“GH”) secretion has been shown to decline during aging, and studies have indicated that GH alters plasma cholesterol (PC) concentrations. For example, a study was conducted to determine how GH secretagogues affect age-related hypercholesterolemia (Walker et al., 1994). In this study, animals were co-administered (s.c.) growth hormone releasing hormone (“GHRH”) and GH-releasing hexapeptide. This study showed that aging was associated with a progressive increase in PC, which was reduced in animals administered GHRH and GHRP compared to those administered vehicle. The results suggest that reduced GH secretion during aging contributes to a progressive increase in plasma cholesterol that can be partially prevented with GH-secretagogues.
Additionally, both growth hormone (“GH”) and insulin-like growth factor I (IGF-I) are involved in heart development and in maintenance of cardiac structure and performance. Cardiovascular disease has been reported to reduce life expectancy in both GH deficiency (“GHD”) and GH excess. Patients with GHD suffer from a cluster of abnormalities associated with increased cardiovascular risk, including abnormal body composition, unfavorable lipid profile, increased fibrinogen and C-reactive protein levels, insulin resistance, early atherosclerosis and endothelial dysfunction, and impaired left ventricular (LV) performance (i.e., reduced diastolic filling and impaired response to peak exercise). Long-term GH replacement therapy reverses most of these abnormalities. More consistently, OH replacement reduces body fat and visceral adipose tissue, reduces low-density lipoprotein cholesterol and triglyceride levels, and improves endothelial function. GH replacement also reduces intima media thickness at major arteries and improves LV performance, but these results have been observed only in small series of patients treated on a short-term basis. This review discusses the roles of GHD and GH replacement therapy in the development of cardiovascular disease (Colao et al., 2006).
Growth Hormone Releasing Hormone (“GHRH”) and Growth Hormone (“GH”) Axis: To better understand how GHRH plasmid-mediated supplementation can be used as a method to decrease cholesterol levels in a subject, the mechanisms and current understanding of the GHRH axis will be addressed. Although not wanting to be bound by theory, the central role of GH is controlling somatic growth in humans and other vertebrates. The physiologically relevant pathways regulating GH secretion from the pituitary are fairly well known. The GH pathway genes include: (1) ligands, such as GH and insulin-like growth factor I (“IGF-I”); (2) transcription factors such as prophet of pit-1 (prop-1), and pit-1: (3) agonists and antagonists, such as GHRH and somatostatin (“SS”), respectively; and (4) receptors, such as GHRH receptor (“GHRH-R”) and the GH receptor (“GH-R”).
These genes are expressed in different organs and tissues, including the hypothalamus, pituitary, liver, and bone. Effective and regulated expression of the GH pathway is essential for optimal linear growth, as well as homeostasis. GH synthesis and secretion from the anterior pituitary is stimulated by GHRH and inhibited by somatostatin, both hypothalamic hormones. GH increases production of IGF-I, primarily in the liver, and other target organs. IGF-I and GH, in turn, feedback on the hypothalamus and pituitary to inhibit GHRH and GH release. GH elicits both direct and indirect actions on peripheral tissues, the indirect effects being mediated mainly by IGF-I.
Supplementation of endogenous GH with injections of recombinant GH peptide has been demonstrated to improve linear growth and/or lean body mass accretion in both animals (Chung et al., 1985; Etherton et al., 1987) and humans (Boguszewski et al., 2005; Lissett and Shalet, 2000). Although this practice is effective, its prolonged use has been linked to some adverse effects including impaired glucose tolerance (Bramnert et al., 2003), fluid retention (Verhelst et al., 1997), and carpal tunnel syndrome (Cummings and Merriam, 1999; Zachwieja and Yarasheski, 1999).
GH secretagogues, especially GHRH, have been considered as an alternative approach to the use of GH either to promote growth or for treatment of conditions that may benefit from activation of the GH/IGF-I axis (Ehlers, 2001). There are several advantages to the use of GHRH: it can stimulate the pulsatile release of endogenous GH (Clark and Robinson, 1985); the feedback control of endogenous GH (Clark et al., 1988) and IGF-I (Ceda et al., 1987) is preserved over a significant dose range, thereby guarding against imbalances between GH and IGF-I levels; the incidence of adverse effects are uncommon (Duck and Rapaport, 1999). Also, GHRH stimulates all GH isoforms, which have been shown to have differential effects in normal and pathological circumstances (Fujikawa et al., 2000; Wallace et al., 2001). Ghrelin is also able to stimulate GH release through its GH-secretagogue receptor (Sun et al., 2004) and has been shown to have a potent impact on fat metabolism (Dornonville de la et al., 2005) and body composition as a potential treatment for cachexia (Nagaya et al., 2005); nevertheless, ghrelin has been reported to stimulate food intake, increase weight gain, and cause obesity (Dornonville de la et al., 2005).
In animals, the short-term or chronic administration of GHRH, either as the recombinant protein or as an analog, improves growth and carcass quality of swine (Phung et al., 2000; Pommier et al., 1990), and lactation performance in dairy cattle (Auchtung et al., 2001; Dahl et al., 1991), but without incurring the side-effects associated with GH. Therapeutic areas also are being investigated for humans for the improvement in body composition in patients with pathological situations, e.g., cachexia (Kotler, 2000), advanced HIV/AIDS (Mulligan and Schambelan, 2002) or cardiac failure (Colao et al., 1999). However, the short half-life of the GHRH peptide in serum (approximately 6 minutes) necessitates injections 1 to 3 times a day, which renders the use of GHRH peptide impractical, especially for long term applications. Plasmid-based GHRH administration is an alternative approach that eliminates the need for repeated administrations of the GHRH peptide or other GH secretagogues.
Previous studies from our laboratory have demonstrated that injection and electroporation of a single dose of a muscle-specific expression plasmid encoding for a GHRH analog cDNA (HV-GHRH) with a longer half-life effectively increased GH and IGF-I concentrations, and improved feed efficiency and weight gain over a 56-day period in swine (Draghia-Akli et al., 1999). In healthy young dogs (Draghia-Akli et al., 2003a), the treatment increased plasma IGF-I levels, body weight, and improved hematological parameters, while maintaining these parameters within the normal limits. In geriatric and cancer-afflicted dogs, plasmid GHRH corrected anemia and cachexia associated with cancer and its therapies (Draghia-Akli et al., 2002a), improved immune function, quality of life and activity levels and furthermore, these effects were maintained long-term after a single administration of the plasmid (Tone et al., 2004).
Growth Hormone Releasing Hormone versus Growth Hormone or Growth Hormone Releasing Peptides (“GHRP”): GH and GHRH are currently administered therapeutically as recombinant proteins. Levels of total cholesterol, triglyceride, free fatty acid, fibrinogen and plasminogen activator inhibitor-1 are decreasing in some patients with GH-deficiencies or high atherosclerotic risk after GH treatment (Ahn et al., 2006; Bollerslev et al., 2005). On the other hand, ghrelin, an upstream stimulator of GH secretion, increases fat deposition, and is positively correlated with cholesterol levels (Langenberg et al., 2005). The effects of GHRH as an unique treatment on cholesterol levels have been unknown to date.
Current knowledge about the interaction between GH and its receptor suggests that the molecular heterogeneity of circulating GH may have important homeostasis implications. It has been suggested that adverse effects including insulin resistance, may result from the fact that exogenous OH elevates the basal GH serum levels and abolishes the natural GH episodic pulses. Studies have shown that continuous infusion with GHRH restores normal GH pulsatile pattern, without desensitization of GHRH receptors or depletion of GH supplies in humans, sheep or pigs (Dubreuil et al., 1990; Vance et al., 1985; Vance et al., 1989). At the same time, this system is capable of feed-back, which is totally abolished in the GH therapies. Virtually no side effects have been reported for GHRH therapies (Thorner et al., 1986a). Thus, GHRH therapy may be more physiological than GH therapy.
GHRPs are used in clinics to stimulate short term GH and IGF-I in humans. Hexarelin, a potent and well-studied GHRP, is capable of causing profound GH release in normal individuals. The GH response to hexarelin in humans becomes appreciably attenuated following long-term administration. Although this attenuation is partial and reversible, it could seriously limit the potential long-term therapeutic use of hexarelin and similar agents (Rahim and Shalet, 1998). With the development of GH-releasing agents and their use in human subjects, it is clear that these agents are not specific for GH release. More recent studies in humans have demonstrated that acute increases in adrenocorticotrophic hormone (ACTH) (Ghigo et al., 1999), cortisol and prolactin (PRL) (Svensson and Bengtsson, 1999) have occurred after administration of GHRPs (hexarelin, MK-0677) (Schleim et al., 1999). The potential adverse effects of repeated episodes of transient (even minor) hyperprolactinaemia and hypercortisolaemia during long-term therapy with GHRPs and similar agents raise concern, require further study, and are undesirable in patients with high cholesterol levels.
In contrast, essentially no side effects have been reported for recombinant GHRH therapies. Extracranially secreted GHRH, as mature peptide or truncated molecules (as seen with pancreatic islet cell tumors and variously located carcinoids) are often biologically active and can even produce acromegaly (Esch et al., 1982; Thorner et al., 1984), when the overexpression persists for more than 7 years. Although recombinant GHRH protein therapy entrains and stimulates normal cyclical GH secretion with virtually no side effects, the short half-life of GHRH in vivo requires frequent (one to three times a day) intravenous, subcutaneous or intranasal (requiring 300-fold higher dose) administration. Thus, as a chronic treatment, GHRH administration is not practical.
Transgene Delivery and in vivo Expression: Although not wanting to be bound by theory, the delivery of a specific transgene to somatic tissue to correct inborn or acquired deficiencies and imbalances is possible. Such transgene-based delivery offers a number of advantages over the administration of recombinant proteins. These advantages include: the conservation of native protein structure; improved biological activity; avoidance of systemic toxicities; and avoidance of infectious and toxic impurities. Because the protein is synthesized and secreted continuously into the circulation by the subject's own cells, plasmid-mediated therapy allows for prolonged production of the protein in a therapeutic range. In contrast, the primary limitation of using recombinant protein is the limited bio-availability of protein after each administration.
A non-viral, plasmid-based expression system, may comprise of a synthetic transgene delivery system in addition to the nucleic acid encoding the therapeutic genetic product. In this way, the risks associated with the use of most viral vectors can be avoided, including the expression of viral proteins that can induce immune responses against the target tissues or the viral vector and the possibility of DNA mutations or activations of oncogenes. The non-viral expression vector products generally have low toxicity due to the use of “species-specific” components for gene delivery, which minimizes the risks of plasmid-targeted immunogenicity and loss of expression. Additionally, no significant integration of plasmid sequences above the rate of spontaneous mutation into host chromosomes has been reported in vivo to date, so that this type of therapy should neither activate oncogenes nor inactivate tumor suppressor genes. As episomal systems residing outside the chromosomes, plasmids have defined pharmacokinetics and elimination profiles, leading to a finite duration of gene expression in target tissues. Plasmid vectors are simple to manufacture using good manufacturing practice techniques. They have a low risk to benefit ratio when compared to viral vectors, as stated on Mar. 13-14, 2003 in a workshop sponsored by the American Society of Gene Therapy (ASGT) and the Food and Drug Administration's Center for Biologics Evaluation and Research (FDA/CBER) (Frederickson et al., 2003).
Direct plasmid DNA gene transfer is currently the basis of many emerging nucleic acid therapy strategies and does not require viral components or lipid particles (Aihara and Miyazaki, 1998; Muramatsu et al., 2001). Skeletal muscle is a preferred target tissue, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that are expressed in immunocompetent hosts (Davis et al., 1993; Tripathy et al., 1996). Plasmid DNA constructs are attractive candidates for direct therapy into the subjects skeletal muscle because the constructs are well-defined entities that are biochemically stable and have been used successfully for many years (Acsadi et al., 1991; Wolff et al., 1990). The relatively low expression levels of an encoded product that are achieved after direct plasmid DNA injection are sometimes sufficient to indicate bio-activity of secreted peptides (Danko and Wolff, 1994; Tsurumi et al., 1996). Our previous reports in mice showed that a human GHRH cDNA could be delivered by direct injection into the muscle by a plasmid where it transiently stimulated GH secretion to a modest extent over a short period (Draghia-Akli et al., 1997).
Plasmid delivery and electroporation: Efforts have been made to enhance the delivery of plasmid DNA to cells by physical means including electroporation, sonoporation, and hydrodynamic pressure. In various tissues, transfection has been enhanced or accomplished by: 1) “gene gun” delivery (usually DNA-coated gold particles propelled into cells); 2) jet injection of DNA (e.g., Biojector); 3) hydrodynamic (intravascular) methods; and 4) by cationic agents such as linear or branched polymers (e.g., polyethylenimines [PEIs]) or cationic liposomes (Akhtar, 2005; El-Aneed, 2004; Patil et al., 2005; Wells, 2004). These methods have their own drawbacks. Gene gun delivery is limited to exposed tissues, intravascular methods often require injection of large volumes of fluid that are not applicable to humans, while complexes of DNA and cationic lipids or polymers can be unstable, inflammatory and even toxic. One of the most versatile and efficient methods of enhancing gene transfer involves the application of electric field pulses after the injection of nucleic acids (DNA, RNA and/or oligonucleotides) into tissues. Although not wanting to be bound by theory, the administration of a nucleic acid construct by electroporation involves the application of a pulsed electric field to create transient pores in the cellular membrane without causing permanent damage to the cell, which allows exogenous molecules to enter the cell (Prud'homme et al., 2006). Nucleic acid molecules may travel through passageways or pores in the cell that are created during the procedure. U.S. Pat. No. 5,704,908 titled “Electroporation and iontophoresis catheter with porous balloon,” issued on Jan. 6, 1998 with Hofmann et al., listed as inventors describes an constant voltage electroporation apparatus for delivering molecules to cells at a selected location within a cavity in the body of a patient. Similar pulse voltage injection devices are also described in: U.S. Pat. No. 5,702,359 titled “Needle electrodes for mediated delivery of drugs and genes,” issued on Dec. 30, 1997, with Hofmann, et al., listed as inventors; U.S. Pat. No. 5,439,440 titled “Electroporation system with voltage control feedback for clinical applications,” issued on Aug. 8, 1995 with Hofmann listed as inventor; PCT application WO/96/12520 titled “Electroporetic Gene and Drug Therapy by Induced Electric Fields,” published on May 5, 1996 with Hofmann et al., listed as inventors; PCT application WO/96/12006 titled “Flow Through Electroporation Apparatus and Method,” published on Apr. 25, 1996 with Hofmann et al., listed as inventors; PCT application WO/95/19805 titled “Electroporation and Iontophoresis Apparatus and Method For insertion of Drugs and genes into Cells,” published on Jul. 27, 1995 with Hofmann listed as inventor; and PCT application WO/97/07826 titled “In Vivo Electroporation of Cells,” published on Mar. 6, 1997, with Nicolau et al., listed as inventors, the entire content of each of the above listed references is hereby incorporated by reference.
Electroporation has been used very successfully to transfect tumor cells after injection of plasmid (Lucas et al., 2002; Matsubara et al., 2001) or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors in humans (Gehl et al., 1998; Heller et al., 1996). Electroporation also has been extensively used in mice (Lesbordes et al., 2002; Lucas et al., 2001; Vilquin et al., 2001), rats (Terada et al., 2001; Yasui et al., 2001), and dogs (Fewell et al., 2001) to deliver therapeutic genes that encode for a variety of hormones, cytokines or enzymes. Previous studies using GHRH showed that plasmid therapy with electroporation is scalable and represents a promising approach to induce production and regulated secretion of proteins in large animals and humans (Draghia-Akli et al., 1999; Draghia-Akli et al., 2002c). Intramuscular injection of plasmid followed by electroporation has been used successfully in ruminants for vaccination purposes (Babiuk et al., 2003; Tollefsen et al., 2003). It has been observed that the electrode configuration affects the electric field distribution, and subsequent results (Gehl et al., 1999; Miklavcic et al., 1998). Although not wanting to be bound by theory, needle electrodes give consistently better results than external caliper electrodes in a large animal model, and can be used for humans. U.S. Pat. No. 4,956,288 is directed to methods for preparing recombinant host cells containing high copy number of a foreign DNA by electroporating a population of cells in the presence of the foreign DNA, culturing the cells, and killing the cells having a low copy number of the foreign DNA.
Constant current versus constant voltage electroporation and species differences: To better understand the process of electroporation, it is important to look at some simple equations. When a potential difference (voltage) is applied across the electrodes implanted in a tissue, it generates an electric field (“E”), which is the applied voltage (“V”) divided by the distance (“d”) between the electrodes: E=V/d
The electric field intensity E has been a very important value when formulating electroporation protocols for the delivery of a drug or macromolecule into the cell of the subject. Accordingly, it is possible to calculate any electric field intensity for a variety of protocols by applying a pulse of predetermined voltage that is proportional to the distance between electrodes. The flow of electric charge (current) between electrodes is achieved by the buffer ions in the tissues, which can vary among tissues and patients. Furthermore, the flow of conducting ions can change between electrodes from the beginning of the electric pulse to the end of the electric pulse. When tissues have a small proportion conducting ions, resistance is increased, heat is generated and cells are killed. Ohm's law expresses the relationship between current (“I”), voltage (“V”), and resistance (“R”): R=V/I
Heating is the product of the inter-electrode impedance (i.e. combination of resistance and reactance and is measured in ohms), and is proportional to the product of the current, voltage and pulse duration. Heating can also be expressed as the square of the current, and pulse duration (“t”, time). For example, during electroporation the heating or power (“W”, watts) generated in the supporting tissue can be represented by the following equation; W=I2Rt.
During pulses, specific tissue resistance may drop (Zampaglione et al., 2005), and the same voltage which did not cause significant heating during the first pulse can burn the tissue during the second (the equation W=V2t/R illustrates this undesirable effect). Constant current EP prevents this overheating, but constant-voltage techniques do not take into account the individual and changing resistance of the tissue and can result in tissue damage, inflammation, and loss of plasmid expression. Recently, we have used instead constant current EP, which we refer to as electrokinetic enhancement. Thus, we have used a software-driven constant-current electroporator denoted electrokinetic device (EKD device) to deliver plasmids to small and large animals (Brown et al., 2004; Draghia-Akli and Fiorotto, 2004; Khan et al., 2005). The most favorable conditions of electroporation were dependent on the individual tissue resistance, which varies by age and species. We found that EP-induced tissue injury can be reduced or eliminated by applying optimal constant current instead of constant voltage. Indeed, this prevents tissue heating and cell death, such as frequently occurs with constant voltage technology and is most relevant to gene therapy in large animals.
The ability of electroporation to enhance plasmid uptake into the skeletal muscle has been well documented. Similarly, plasmids formulated with poly-L-glutamate (“PLG”) or polyvinylpyrrolidone (“PVP”) were observed to have an increase in plasmid transfection, which consequently increased the expression of a desired transgene. For example, plasmids formulated with PLG or PVP were observed to increase gene expression to up to 10 fold in the skeletal muscle of mice, rats, and dogs (Fewell et al., 2001; Mumper et al., 1998). Nevertheless, in these cases, expression was short lived and correlated with tissue damage. Although not wanting to be bound by theory, the anionic polymer sodium PLG enhances plasmid uptake at low plasmid concentrations and may reduce any possible tissue damage caused by the procedure if of a certain molecular weight and concentration (Draghia-Akli et al., 2002b). PLG is a stable compound and it is resistant to relatively high temperatures (Dolnik et al., 1993). PLG has been used to increase stability of anti-cancer drugs (Li et al., 2000) and as “glue” to close wounds or to prevent bleeding from tissues during wound and tissue repair (Otani et al., 1996; Otani et al., 1998). PLG also has been used as an anti-toxin after antigen inhalation or exposure to ozone (Fryer and Jacoby, 1993).
Although not wanting to be bound by theory, we have demonstrated PLG increases the transfection of the plasmid during the electroporation process, not only by stabilizing the plasmid DNA and facilitating the intracellular transport through the membrane pores, but also through an active mechanism. For example, positively charged surface proteins on the cells could complex the negatively charged PLG linked to plasmid DNA through protein-protein interactions. When an electric field is applied, the surface proteins reverse direction and actively internalize the DNA molecules, a process that substantially increases the transfection efficiency. Furthermore, PLG will prevent the muscle damage associated with in vivo plasmid delivery (Draghia-Akli et al., 2002b) and will increase plasmid stability in vitro prior to injection.
Although not wanting to be bound by theory, a GHRH cDNA can be delivered to muscle of mice injectable myogenic expression vector where it can transiently stimulate GH secretion over a period of two weeks (Draghia-Akli et al., 1997). This injectable vector system was optimized by incorporating a powerful synthetic muscle promoter (Li et al., 1999) coupled with a novel protease-resistant GHRH molecule with a substantially longer half-life and greater GH secretory activity (pSP-HV-GHRH) (Draghia-Akli et al., 1999). Highly efficient electroporation was optimized to deliver the nucleic acid construct to the skeletal muscle of an animal (Prud'homme et al., 2006). Using this combination of vector design and electric pulses plasmid delivery method, the inventors were able to show increased growth and favorably modified body composition in pigs (Draghia-Akli et al., 1999; Draghia-Akli et al., 2003b). The modified GHRH nucleic acid constructs increased red blood cell production in companion animals with cancer and cancer treatment-associated anemia (Draghia-Akli et al., 2002a). In pigs, available data suggested that the modified porcine HV-GHRH analog (SEQ ID#1) was more potent in promoting growth and positive body composition changes than the wild-type porcine GHRH (Draghia-Akli et al., 1999).
Administering novel GHRH analog proteins (U.S. Pat. Nos. 5,847,066; 5846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505; 5,137,872; 5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512, RE33,699) or synthetic or naturally occurring peptide fragments of GHRH (U.S. Pat. Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857; 4,224,316; 4,223,021; 4,223,020; 4,223,019) for the purpose of increasing release of growth hormone have been reported. A GHRH analog containing the following mutations has been reported (U.S. Pat. No. 5,846,936): Tyr at position 1 to His; Ala at position 2 to Val, Leu, or others; Asn at position 8 to Gln, Ser, or Thr; Gly at position 15 to Ala or Leu; Met at position 27 to Nle or Leu; and Ser at position 28 to Asn. The GHRH analog is the subject of U.S. Pat. No. 6,551,996 titled “Super-active porcine growth hormone releasing hormone analog,” issued on Apr. 22, 2003 with Schwartz, et al., listed as inventors (“the '996 patent”), which teaches application of a GHRH analog containing mutations that improve the ability to elicit the release of growth hormone. In addition, the '996 patent application relates to the treatment of growth deficiencies; the improvement of growth performance; the stimulation of production of growth hormone in an animal at a greater level than that associated with normal growth; and the enhancement of growth utilizing the administration of growth hormone releasing hormone analog and is herein incorporated by reference.
In summary, decreasing the cholesterol levels in a subject was previously uneconomical and restricted in scope. The related art has shown that it is possible to impact this condition in a limited capacity utilizing recombinant protein technology, but these treatments have some significant drawbacks. It has also been taught that nucleic acid expression constructs that encode recombinant proteins are viable solutions to the problems of frequent injections and high cost of traditional recombinant therapy. There is a need in the art to expanded treatments for subjects with a disease by utilizing nucleic acid expression constructs that are delivered into a subject and express stable therapeutic proteins in vivo.